This invention relates generally to pro-apoptotic genes and gene products and, more specifically, to a member of the Bcl-2 gene family.
Tissue homeostasis is maintained by the process of apoptosisxe2x80x94that is, the normal physiological process of programmed cell death. Changes to the apoptotic pathway that prevent or delay normal cell turnover can be just as important in the pathogenesis of diseases as are abnormalities in the regulation of the cell cycle. Like cell division, which is controlled through complex interactions between cell cycle regulatory proteins, apoptosis is similarly regulated under normal circumstances by the interaction of gene products that either prevent or induce cell death.
Since apoptosis functions in maintaining tissue homeostasis in a range of physiological processes such as embryonic development, immune cell regulation and normal cellular turnover, the dysfunction or loss of regulated apoptosis can lead to a variety of pathological disease states. For example, the loss of apoptosis can lead to the pathological accumulation of self-reactive lymphocytes that occurs with many autoimmune diseases. Inappropriate loss or inhibition of apoptosis can also lead to the accumulation of virally infected cells and of hyperproliferative cells such as neoplastic or tumor cells. Similarly, the inappropriate activation of apoptosis can also contribute to a variety of pathological disease states including, for example, acquired immunodeficiency syndrome (AIDS), neurodegenerative diseases and ischemic injury. Treatments which are specifically designed to modulate the apoptotic pathways in these and other pathological conditions can alter the natural progression of many of these diseases.
Although apoptosis is mediated by diverse signals and complex interactions of cellular gene products, the results of these interactions ultimately feed into a cell death pathway that is evolutionarily conserved between humans and invertebrates. The pathway, itself, is a cascade of proteolytic events analogous to that of the blood coagulation cascade.
Several gene families and products that modulate the apoptotic process have now been identified. One family is the Bcl-2 family. Bcl-2 is the first recognized component of programmed cell death or apoptosis (Tsujimoto et al., Science 228: 1440-1443, 1985; Nunez, et al., J. Immunol. 144: 3602-3610, 1990), an evolutionary conserved process essential for normal development of multicellular organisms and in maintaining tissue homeostasis. Various aspects of involvement of Bcl-2 in this process have been well documented at the molecular and physiological levels (for review see Reed, Nature 387: 773, 1997). Bcl-2 is a multifunctional 239 amino acid protein that has a hydrophobic C-terminal membrane anchor preceded by three domains designated BH1, BH2 and BH3 (Bcl-2 Homology domains 1, 2 and 3) that are necessary for its function. Bcl-2 and its homologue Bcl-xL are death antagonists that associate mainly with the outer mitochondrial membrane, the endoplasmic reticulum, and nuclear envelope and moreover, have documented ion channel activity (Reed, Nature 387: 773, 1997). These proteins may prevent apoptosis by regulating the electrical and osmotic homeostasis of the mitochondria, a process that is required to prevent mitochondrial swelling, outer membrane rupture and cytochrome c release (Vander Heiden et al., Cell 91: 627-637, 1997). Cytochrome c release from the mitochondria is believed to trigger activation of the death caspase cascade, through formation of the Apaf-1/caspase-9/cytochrome c complex (Li et al., Cell 91: 479-489, 1997; Reed, Cell 91: 559-562, 1997).
Intriguingly, among the members of the Bcl-2 family of proteins discovered in recent years there are death agonists (e.g. Bax, Bad, Bik, Bak, Bid and Hrk). Except for Bad whose BH3 domain is within the putative BH1 domain, all of these proteins contain an independent BH3 domain (Zha, et al., J. Biol. Chem. 272: 24101-24104, 1997). It appears that only the BH3 domain is required for their pro-apoptotic activity (Wang, et al., Genes Dev. 10: 2859-2869, 1996; Inohara, et al., EMBO J. 16: 1686-1694, 1997; Zha, et al., J. Biol. Chem. 272: 24101-24104, 1997; Chittenden et al., EMBO J 14: 5589-5596, 1995; Boyd, et al., Oncogene 11: 1921-1928, 1995; Zha, et al., J. Biol. Chem. 271: 7440-7444, 1997; Hunter and Parslow, J. Biol. Chem. 271: 8521-8524, 1996; Sattler, et al., Science 275: 1129-1132, 1997). This domain interacts with a hydrophobic cleft formed by the BH1, BH2 and BH3 domains of the anti-apoptotic Bcl-xL and Bcl-2 as evident from mutational and structural studies (Sattler, et al., Id,). Interestingly, Bik, Bid and Hrk, which contain only a BH3 domain, seem more potent death effectors than those proteins with all three domains (Bax and Bak) (see Wang, et al., Genes Dev. 10: 2859-2869, 1996).
Therefore, there exists a need in the art for methods of assaying compounds for their ability to affect binding activity of the pro-apoptotic with the anti-apoptotic Bcl-2 family members as well as for methods of regulating these proteins in order to treat diseases and syndromes. The present invention provides recombinant Blk constructs that are active in cells, allowing the regulation of apoptosis for the treatment of pathology as well as providing methods and compositions for assaying compounds that inhibit binding of Blk to anti-apoptotic proteins, while further providing other related advantages.
As noted above, the present invention provides recombinant Blk constructs, mammalian Blk proteins, antibodies that selectively bind to such proteins, and related pharmaceutical compositions and methods. In particular, in one aspect of the invention, nucleic acid molecules encoding mammalian Blk, including human or murine Blk, are provided. Within certain embodiments, the mammalian Blk has the amino acid sequence of SEQ ID No. 2, or a variant thereof, or is encoded by the nucleotide sequence of SEQ ID No. 1 or a variant thereof. In other related aspects, the invention provides a nucleotide sequence encoding a BH3 domain of a mammalian Blk protein. In one embodiment, the BH3 domain has the amino acid sequence Ala Leu Arg Leu Ala Cys Ile Gly Asp Glu Met Asp (SEQ ID NO:3), optionally including one or more amino acid substitutions. Nucleic acid vectors comprising such Blk sequences and host cells containing such vectors are also provided.
Within another aspect of the invention, isolated mammalian Blk proteins, as well as an isolated BH3 domain of a mammalian Blk protein, are provided. Within a related aspect, antibodies that selectively bind to a mammalian Blk protein are disclosed. In one embodiment, the antibody inhibits the binding of Blk to either Bcl-2 or Bcl-xL. The antibodies provided herein include antibody fragments, single chain antibodies, and humanized antibodies.
Within still other aspects of the subject invention, pharmaceutical compositions comprising the mammalian Blk proteins and gene delivery vehicles comprising nucleic acid molecules as described above are provided.
Within other aspects, methods for treating unwanted cell proliferation through the use of effector molecules that increase unbound Blk protein in a cell are provided. Within certain embodiments, the effector molecule is a peptide that comprises residues 52 to 63 of SEQ ID No. 2 or a variant thereof, wherein the variant has one of more amino acid substitutions at a residue other than Leu55, Gly59 or Asp60. Gene delivery vehicles containing a nucleic acid molecule encoding such a peptide may also be used.
Within a related aspect, methods are provided for screening for such effector molecules, comprising contacting a candidate effector molecule with a solution comprising Blk and Bcl-2 or Bcl-xL; and detecting unbound Blk, wherein an increase in the amount of unbound Blk in the presence of the candidate effector molecule (as compared to the absence of the molecule) indicates that the candidate molecule is suitable for treating unwanted cell proliferation through increasing unbound Blk protein.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references that are set forth below describe in more detail certain procedures or compositions and are therefore incorporated by reference in their entirety.